Recent Research in Science and Technology 2010, 2(6): 110-115

ISSN: 2076-5061
www.recent-science.com
BOTANY

ARACHIS BIOASSAY FOR SOIL CONTAMINATED WITH HEXAVALENT

CHROMIUM
K. Rajalakshmi1, P. Kumar2, A. Saravanakumar2, A. Aslam2, A. Shahjahan2 and R. Ravikumar2
1Department
2PG

of Botany, NGM College, Pollachi 642 001, Coimbatore, Tamilnadu, India

and Research Department of Botany, Jamal Mohamad College, Tiruchirappalli 620020, Tamilnadu, India

Abstract

Heavy metals evoke multiple direct and indirect effects on plant growth and affect many physiological functions. Negative
impacts include inhibition of seed germination, reduction in plant growth and yield and metabolic disturbances evaluated in
terms of altered biochemicals. In vitro and pot culture studies revealed that there is a significant uptake of Chromium by
natural growth conditions. There was also a strong negative correlation between the concentration of Cr(VI) and the biomass
production in A. hypogea. The control plant grows tall than any other Chromium treated plant. The paper wig method
(Whattman filter paper number 3) was found to be effective in screening the Cr(VI) absorption. Interestingly, Chromium in mild
concentrations is promoting the cellular elongation resulting in the longest root of seedlings reared in the 1mM hexavalent
Chromium. Chromium when supplemented with the selected bioinoculants to the experimental formulations showed promising
results in the plant growth and development than the control seedlings. Both pot cultured plants and in vitro raised plants were
found to contain 375.80 ppm (per 5g) and 47.06 ppm (per 5g) of Chromium respectively in the Atomic Absortion Spectrometric
quantifications. Further experiments are underway to study the biological effects and accumulation of absorbed Cr(VI) in this
economically important legume.
Keywords: Chromium, Cr(VI), Heavy metal toxicity, Bioaccumulation, Arachis hypogea

Introduction

The ubiquity of heavy metals in the environment

results in the introduction of high amounts of toxic
metals into the food chain from various sources. The
heavy metals commonly found in the environment
include Cu, Zn, Ni, Pb, Cd, Co, Hg, Cr and As. Some
of these metals act as micronutrients at small
concentration in living organism for their normal
physiological activities; however accumulation is toxic
to most life forms. The toxicity of heavy metals is
mainly attributed to their ability in binding with enzymes
which in turn brings forth alteration of catalytic
functions and inactivation [1, 2]. Heavy metals also
bind strongly to oxygen, nitrogen and sulphur atoms
that often interrupt the spatial configuration of
biomolecules [3].
Chromium is used in several industries such as
metal finishing, petroleum refining, iron and steel
industries, leather tanning, inorganic chemicals
production, textile manufacturing and pulp producing,
electroplating and mine tailings [4]. The leather industry
is the major cause for the high influx of chromium to
the biosphere, accounting for 40% of the total industrial
use [4]. In India, about 2000 to 32,000 tons of
elemental chromium annually escapes into the
environment from tanning industries [5]. The toxicity of
chromium is highly dependent on its oxidation state.

Corresponding Author, Email: ecoraji@gmail.com

Cr(III) species are less toxic and less mobile, with very
low solubility at all pH levels above 5.5 [5].
Reports are available on inhibitory effects of
Chromium on growth and metabolism of many plant
species like mosses, rice, pea, wheat, etc. in relation to
oxidative stress. Accumulation of Cr(VI) by plants can
reduce growth, induce chlorosis in young leaves,
reduce pigment content, alter enzymatic function,
damage root cells and cause ultrastructural
modifications of the chloroplast and cell membrane [6,
7, 8, 9,10]. Roots accumulate several magnitudes
higher chromium than shoots. The excess soluble
salts in the root causes osmotic stress resulting in the
disturbance of the plant water relation, uptake and
utilization of essential nutrients. At the cellular level,
both Cr(VI) and Cr(III) are toxic to plants. Cr(VI) is a
strong oxidizing agent and causes severe damage to
cell membranes [11]. Chromium (VI) is toxic to plants
because of its ability to form complexes with nucleic
acids, proteins and organic compounds.
In vitro trials offer a wide scope in understanding
the mechanism and dynamics of plant growth and
development as we can control, examine and
manipulate the desirable variables involved. Literature
is available for different scientific enquiries of
developmental, physiological, biochemical and
molecular evidences have been carried out in vitro.
This present study is an earnest attempt to investigate

K. Rajalakshmi et al./Rec Res Sci Tech 2 (2010) 110-115

using standard procedure. Dried soil samples were

homogenized with clean dry pestle and mortar. Then
the samples were sieved with a metal mesh of ~2mm
size. The following formulations were used to fill paper
cups of 100 ml capacity to raise A. hypogea seedlings.
The soil samples were collected from three sites viz.,
soil from agriculture land near the effluent stream (Soil
1), bottom soil of the effluent stream (Soil 2), and
Garden soil with rich compost (Soil 3). The Table 1
indicates the various formulations of the experimental
soil mixtures and their ratios. Commercially available
bioinoculants were mixed in different combinations.

the biological effects of Cr (VI) on plants of A. hypogea,

in vitro and in vivo and aims to evaluate the
morphogenetic changes during seedling growth to
predict the levels of Cr(VI) in aqueous extracts of soil
polluted with hexavalent chromium.

Materials and Methods

Screening of Seedling growth in tannery effluent

soil
Soils were collected from tannery effluent infested
fields in the environs of Shanmuga tanning factory,
Gundur, Tiruchrappalli district. The soil samples were
dried in hot air oven at 80oC for 48 hrs then tyntallized

Table 1. Soil mixtures and their ratios

S.No.

Experiment code

Soil 1 (g)

Soil 2 (g)

Soil 3 (g)

Bioinoculant (g)

1.
2.

Exp-1
Exp-2

0
25

100
75

3.

Exp-3

50

50

4.

Exp-4

75

25

5.

Exp-5

100

6.

Exp-6

100

7.

Exp-7

25

75

8.

Exp-8

50

50

9.

Exp-9

75

25

10.

Exp-10

100

11.

Exp-11

100

12.

Exp-12

25

75

13.

Exp-13

50

50

14.

Exp-14

75

25

15.

Exp-15

100

16.

Exp-16

100

17.

Exp-17

25

75

10

18.

Exp-18

50

50

10

19.

Exp-19

75

25

10

20.

Exp-20

100

10

21.

Exp-21

100

10

22.

Exp-22

25

75

10

23.

Exp-23

50

50

10

24.

Exp-24

75

25

10

25.

Exp-25

100

10

26.

Exp-26

100

10

27.

Exp-27

25

75

10

28.

Exp-28

50

50

10

29.

Exp-29

75

25

10

30.

Exp-30

100

10

After 40 days of seedling growth both in vitro and

paper cups they were plucked out and washed in
running tap water and distilled water. Then air dried

and then oven dried for 48 hrs at 80oC. The powder

was digested with concentrated HNO3. Heavy metal
analysis was carried out for the quantification of Cr(VI)

K. Rajalakshmi et al./Rec Res Sci Tech 2 (2010) 110-115

and an 18 hrs illumination of 1000 lux intensity was

provided to cultures with cool, white fluorescent lamps.

by Atomic Absorption Spectrophotometer (Analyst

400/HGA900/AS800 Perkin Elmer) at Centre for
Advanced Research in Indian System of Medicine
(CARISM), SASTRA University, Thanjavur 613 402.

Results

Both the root system and shoot system responded

to the gradient of the Cr(VI) concentrations. There was
a strong negative correlation between the
concentration of Cr(VI) and the biomass production in
A. hypogea seedlings. The paper wig method was
effective in screening the Cr(VI) absorption. There was
a visual evidence of the color change in the paper wig
evidenced by the capillary movement. This proves that
the control plant obtained the nutrients in the same way.
The paper wig was strong only when prepared with
Whattman filter paper number 3 (Plate 2). The plant
height is negatively correlated with the concentration of
the Cr(VI) as evidenced by the Table-1. Both root
length and shoot length and root length are found to be
affected by the increasing concentrations of Cr(VI).

In vitro assay for chromium toxicity

Fruits of Arachis hypogea L. cv VR-2 procured
from Anbil Dharmalingam College of Agriculture,
Tiruchirappalli were used as explant. MS liquid medium
(half strength) was used for the present work.
Inoculation was carried out aseptically. A stock solution
of 0.5M was prepared. By dilution of 0.5 M stock,
different levels of chromium concentration (0.5, 1, 2,
3and 5mM) had been incorporated into half strength
MS liquid medium under aseptic conditions. Seeds
were inoculated over a M shaped paper wig made by
sterilized Whatman No2 filter paper (Figure 1). After
inoculation, cultures were moved to the incubation
room, where the temperature hovered around 2520c
Figure 1

K. Rajalakshmi et al./Rec Res Sci Tech 2 (2010) 110-115

The high Cr(VI) concentrations not only affect the

terminal bud growth and development but also the
nodal branch formation as well the endogenously
originated secondary branches of roots. This is
evidenced by the reduced number of branches and
number of leaves in Table 1. The cellular elongation in
the roots is responsible for the length of roots. Mild
concentrations like 1 mM Cr(VI) promotes the cellular
elongation resulting in the longest root of seedlings.
Regarding the fresh weight of the shoots there is a
normal distribution of fresh weight in correlation with

the increasing Cr(VI). Pot culture studies revealed that

there is significant uptake of the Chromium by natural
growth conditions also. In comparison with the
experimental formulations without bio-inoculants, the
experimental formulations with bioinoculants showed
efficient growth.
Regarding the Chromium
accumulation in plants, both the in vitro and pot
cultured plants showed 375.80 ppm and 47.06 ppm
respectively
in
the
Atomic
Absorption
Spectrophotometer quantifications.
Figure 2

K. Rajalakshmi et al./Rec Res Sci Tech 2 (2010) 110-115

Table 2 Chromium inflicted morphometric characters in in vitro and pot cultures of
Arachis hypogea L.
Parameters
Plant total
height (cm)
Root length
(cm)
Shoot
length (cm)
No. of
branches
No. of
leaves
No. of
branched
root
Longest
branched
root (cm)
Shoot fresh
weight (g)
Root fresh
weight (g)
Max. leaf
surface
area (sq
cm)
Min. leaf
surface
area (sq
cm)

Control
F
I

1mM
F

2mM
F

4mM
F

6mM
F

8mM
F

10mM
F
I

12mM
F
I

20.1

17.9

12.5

15.2

11.2

13.1

8.8

6.4

8.2

6.3

8.0

5.4

7.7

4.3

7.7

3.2

9.3

8.4

2.9

4.4

2.8

3.2

2.1

1.6

2.0

1.2

1.5

0.8

1.5

0.6

1.2

0.4

10.8

9.5

9.6

10.2

8.4

9.8

6.7

5.4

6.2

4.3

6.5

3.4

5.2

2.2

5.5

1.6

24

16

12

16

12

16

12

11

12

12

12

12

30

26

22

28

23

25

20

22

21

18

23

16

26

14

16

12

1.2

0.9

5.0

3.0

2.9

4.1

1.7

1.4

2.0

1.6

1.7

1.2

1.2

0.8

1.3

0.4

0.764

0.612

0.629

0.812

0.684

0.801

0.659

0.541

0.664

0.521

0.591

0.042

0.427

0.312

0.464

0.210

0.063

0.047

0.059

0.041

0.055

0.041

0.052

0.041

0.046

0.031

0.042

0.031

0.026

0.012

0.018

1.8

1.4

0.7

0.9

0.6

0.5

0.8

0.7

0.7

0.5

0.6

0.4

0.6

0.3

0.4

0.2

0.3

0.3

0.24

0.41

0.15

0.21

0.4

0.3

0.4

0.3

0.24

0.1

0.35

0.15

0.12

0.12

F Field grown plant ; I In vitro reared plant

Figure 3 Chromium inflicted morphometric characters in vitro cultures of A. hypogea

Discussion
The present study indicates that the chromium
inhibited the plant growth and development at higher
concentrations (4 mM to 12 mM) both in vitro and in pot
cultures. Chromium toxicity deleteriously affects the
percentage germination, root growth, shoot growth,
that has been widely reported [8, 11]. Seedling
adapted well on half-strength MS medium augmented
with lower chromium concentration (0.5mM & 1.0 mM).
Table 2 reveals the morphometric evaluations such as
percentage germination, root-shoot growth fresh and
dry weight of both root and shoot systems of mild

concentrations of chromium did not deviate much from

the control. Figure 1 (e) shows the chromium in mild
concentrations are promoting the cellular elongation
resulting in the longest root of seedlings raised in the
1mM hexavalent Chromium. The observations are
corroborating with our previously reported [12] fact that
chromium supplemented at extremely low
concentrations even enhanced greening of callus and
culture tissue in selected treatments.
Threshold for inflicting injuiries in field studies and
pot experiments is significantly higher than what is
required for soil less in vitro system [13]. The results of

K. Rajalakshmi et al./Rec Res Sci Tech 2 (2010) 110-115

the present investigation of chromium-invoked inquiries

were comparatively lesser in pot cultures than in in
vitro that accords with the elemental property of Cr(VI)
[14, 15]. And interestingly, the pot cultures
supplemented with bioinoculants showed no significant
growth change even when chromium is presented at
slightly higher concentrations (4 mM to 8 mM) and this
may be due to the intervention of the bioinoculant with
the chromium absorption and transport.
Figure 1(a, b, c, d) presents the novel technique of
employing M shaped paper wig that emerged in this
course of study. The colour development from straw
yellow to orange yellow confirmed the idea of
chromium absorption via capillary movement that
emphasizes the effect of chromium in water relations
and mineral uptake. Turner and Rust in the year 1971
[15] reported wilting of various plant species due to
chromium toxicity that coincides with the present
results. The seedlings both in vitro and pot cultures
showed the signs of wilting that includes drooping leaf
and leaves with minimized surface area. However it
was not true with the pot cultures bestowed with
bioinoculants (Figure 2: f, g, h). Evaluating the effects
of Cr(VI), and the paper wig method provides a new
avenue of screening the cultivar varieties for Cr(VI)
tolerance [16].
This study confirms that in vitro assays are
valuable for its greater flexibility and precision, found
place in many applied frontiers of agriculture,
horticulture and biotechnology. Further experiments
can help to study the biological accumulation of
absorbed Cr(VI) in edible parts of A. hypogea
economically important legume of the tropical world.

5.

6.

7.
8.
9.

10.
11.

12.

13.

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